This project aims to investigate the role that epigenetic silencing ("epimutation") of the DNA mismatch repair (MMR) gene hMLH1 plays in the development of tumors that are deficient in MMR. Hereditary non-polyposis colorectal cancer (HNPCC) is a familial syndrome in which patients develop multiple cancers due to genetic mutation of a MMR gene. But many patients who appear to have HNPCC do not have an identifiable mutation. Epimutation can mimic genetic mutation by inactivating a gene. An epimutation may be present in germline cells, and so be heritable, but it is not passed on in the same way as a change in DNA sequence. We have found that some people with apparent HNPCC carry such an epimutation in hMLH1, which can be identified by assessing cytosine methylation in the hMLH1 promoter. We propose to identify more such cases by screening suspected HNPCC cases who lack mutation in an MMR gene. This strategy may identify [unreadable] kindreds in which the epimutation has been inherited, explaining why in some families cancer predisposition does not behave as a Mendelian trait. We will also ask if epimutation of hMLH1 is more likely to occur on certain genotypes at the hMLH1 locus, which would suggest that something about the genetic architecture of the locus can predispose it to silencing. Many sporadic (non-familial) cases of cancer exhibit deficiency in MMR. We hypothesize that some individuals have an increased risk of developing a cancer because they carry the hMLH1 epimutation in only a small proportion of their cells, i.e., they are mosaic. We will measure the proportion of the hMLH1 epimutation in peripheral blood of 2000 normal blood donors, thus establishing a normal range. We will also assess the proportion of hMLH1 epimutation in patients with sporadic MMRdeficient tumors. These studies have the potential to uncover evidence that epimutation of a tumor suppressor gene is an underlying cause of sporadic cancers. They may also produce the first clear evidence that epigenetic silencing can be inherited to produce a familial disorder such as HNPCC. [unreadable] [unreadable] [unreadable]